The role of human and Drosophila NXF proteins in nuclear mRNA ...

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Introduction 26 experiments revealed that the presence of a poly(A) tail does not significantly enhance the export efficiency of these transcripts (see above; Jarmolowski et al., 1994). Another link between nuclear mRNA export and 3' end formation has been made by analyzing transcripts in yeast strains defective in mRNA export. Defects in RAT7 (Nup159), GLE1, MEX67, RAT8 (Dbp5) or RIP1 genes, which all result in a block to mRNA export, cause hyperadenylation of transcripts (Hammell et al., 2002; Hilleren and Parker, 2001; Jensen et al., 2001b). Furthermore, 3'-extended mRNAs (resulting from improper transcription termination) are frequently observed in mRNA export mutant strains (Hammell et al., 2002). These 3'-extended and/or hyperadenylated mRNAs accumulate at the site of transcription. Similarly, incompletely processed transcripts also accumulate at the site of transcription in mammalian cells (Custodio et al., 1999; Hilleren et al., 2001; Jensen et al., 2001b; Libri et al., 2002). Interestingly, not all yeast mRNA export mutants show defects in 3' end formation: yra1 or sub2 mutants neither display termination nor polyadenylation defects (Hammell et al., 2002). Even though these data indicate a coupling between 3' end formation and mRNA export providing an efficient retention mechanism of incompletely processed transcripts, the molecular basis underlying this phenomenon remains to be established. Other factors implicated in mRNA export Figure 6: mRNA export is coupled to other processes The recruitment of the mRNA export machinery can be influenced by splicing, transcription and 3' end formation. Moreover, a cotranscriptional surveillance mechanism might control mRNA "structure" before export (see also section 3.3). UAP56, NXF1 and p15 are essential for bulk mRNA export, but REF proteins are not (in Drosophila), indicating that other factor(s) must exist which can mediate the recruitment of the NXF1:p15 heterodimer to cellular mRNAs (modified from Cullen, 2003). Apart from NXF proteins and the different factors directly implicated in their recruitment, several other proteins have been suggested to play a role in mRNA export. Amongst many others, these include different nucleoporins and the conserved proteins Gle1, Gle2 and Dbp5.
Introduction 27 In S. cerevisiae, several nucleoporins playing a role in mRNA export have been identified, examples being Nup159p (also called Rat7p), Nup133p (also called Rat3p), Nup116p, Nup85p (also called Rat9p) and Nup145p. In mammals, the nucleoporins Nup98, Nup153, Nup133, Nup160 and TPR have been implicated in this process (reviewed by: Gorlich and Kutay, 1999; Vasu et al., 2001). Inactivation of these nucleoporins does not result in a general block of all nucleocytoplasmic transport processes, but in an mRNA export defect, indicating that they might provide essential docking sites which are preferentially used by the mRNA export machinery. S. cerevisiae Gle1p (also called Rss1p) is an essential protein which is concentrated at the cytoplasmic side of the NPC. Mutant strains display strong defects in mRNA export. Gle1p has been shown to contain a leucine-rich export signal which is not present in the human homologue (Del Priore et al., 1996; Murphy and Wente, 1996; Strahm et al., 1999). Interestingly, human Gle1 can only complement the yeast mutant if a leucine-rich export signal is inserted (Watkins et al., 1998). Human Gle1 is also concentrated at the NPC, and anti-Gle1 antibodies inhibit mRNA export (but not protein import) when injected into HeLa cells, providing evidence for a role in this process (Watkins et al., 1998). However, neither human nor yeast Gle1 has been shown to shuttle or to interact with RNA. While Rae1p is essential in S. pombe, its orthologue Gle2p is not in S. cerevisiae (Brown et al., 1995; Murphy et al., 1996). Mutations in GLE2 or RAE1 cause nuclear accumulation of poly(A) + RNA, and both Gle2p and Rae1p are associated with the NPC. Gle2p was also shown to interact directly with the nucleoporin Nup116p (Bailer et al., 1998; Murphy et al., 1996). Furthermore, both Gle2p and Rae1p interact with Mex67p (Yoon et al., 2000; Zenklusen et al., 2001). Similarly, the human homologue hRAE1 interacts with TAP, shuttles and localizes to the NPC in a transcription-dependent manner (Bachi et al., 2000; Bailer et al., 1998; Pritchard et al., 1999). This association is at least in part mediated by its direct interaction with the nucleoporin Nup98 (Pritchard et al., 1999). hRAE1 was also shown to interact directly or indirectly with mRNPs, as it can be crosslinked to poly(A) + RNA (Kraemer and Blobel, 1997). The conserved interaction between Gle2p/RAE1 and Mex67p/TAP suggests that these proteins might cooperate to export mRNAs, but the precise role of Gle2 in this process remains to be investigated. Dbp5 belongs to the DEAD-box-containing family of RNA helicases. Dbp5 is mainly cytoplasmic, but a fraction of Dbp5 localizes to the cytoplasmic fibrils of the NPC in both yeast and mammalian cells (Schmitt et al., 1999; Snay-Hodge et al., 1998; Tseng et al., 1998). This association with the NPC is mediated through its direct interaction with the nucleoporin CAN/Nup159p (Hodge et al., 1999; Schmitt et al., 1999). Yeast Dbp5p shuttles and dbp5 mutant yeast strains display a block to poly(A) + RNA export (Hodge et al., 1999; Snay-Hodge et al., 1998; Tseng et al., 1998). Similarly, injection of anti-Dbp5
Page 1 and 2: The role of human and Drosophila NX
Page 3 and 4: This thesis describes work carried
Page 5 and 6: Table of Contents Table of Contents
Page 7 and 8: Summary Summary 1 A distinguishing
Page 9 and 10: Summary 3 Crm1 resulted in decrease
Page 11 and 12: Zusammenfassung 5 NXF1 durch RNAi v
Page 13 and 14: 1 Introduction 1.1 Nucleocytoplasmi
Page 15 and 16: 1.1.4 The importin ββ-like family
Page 17 and 18: 1.2 The nuclear export of RNAs Intr
Page 19 and 20: Introduction 13 with the export of
Page 21 and 22: Introduction 15 What are the critic
Page 23 and 24: Introduction 17 mRNA export are ind
Page 25 and 26: Introduction 19 The NPC-binding dom
Page 27 and 28: Introduction 21 Association of the
Page 29 and 30: Introduction 23 could trigger ATP-d
Page 31: Introduction 25 Sub2p and Yra1p and
Page 35 and 36: Introduction 29 export block of bul
Page 37 and 38: Introduction 31 efficiently blocks
Page 39 and 40: 2 Results/Publications Results/Publ
Page 41 and 42: Results/Publications 35 shown to in
Page 43 and 44: VOL. 20, 2000 NXF MULTIGENE FAMILY
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Page 55 and 56: Results/Publications 49 2.1.2 Paper
Page 57 and 58: Research Paper 1381 NXF5, a novel m
Page 59 and 60: Research Paper Loss of the NXF5 gen
Page 63 and 64: Figure 4 Research Paper Loss of the
Page 69 and 70: Results/Publications 63 with the nu
Page 71 and 72: RNA (2001), 7:1768-1780+ Cambridge
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1780 A. Herold et al. Black BE, Hol
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Results/Publications 79 mRNAs had a
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Herold et al. Results/Publications
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Herold et al. (Supplemental Materia
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Herold et al. (Supplemental Materia
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A 10000 reference 1000 100 10 10000
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A 10 1 -10 3.07 (11) 2.37 4.74 (20)
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A B Northern blot dsRNA sd06908: cl
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GFP day4 NXF1 day2 NXF1 day4 p15 da
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Supplemental Table I. mRNAs not aff
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GH06306 1.86 1.49 2.60 1.65 2.95 2.
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Supplemental Table III: mRNAs that
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Supplemental Table IV: mRNAs that a
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2.3 Functional analysis of TAP/NXF1
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THE JOURNAL OF BIOLOGICAL CHEMISTRY
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20538 FIG. 1. Domain organization o
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20540 FIG. 4. TAP stimulates export
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20542 CRM1-mediated export of U snR
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Results/Publications 127 2.3.2 Pape
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MOLECULAR AND CELLULAR BIOLOGY, Aug
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VOL. 22, 2002 p15-INDEPENDENT NUCLE
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Results/Publications 143 2.4 Role o
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The EMBO Journal Vol. 21 No. 20 pp.
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B.M.H.Lange et al. somes at metapha
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B.M.H.Lange et al. Fig. 5. Aurora B
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B.M.H.Lange et al. Fig. 6. Aurora B
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B.M.H.Lange et al. released along t
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B.M.H.Lange et al. protein kinase i
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Discussion 157 strain, already poin
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Discussion 159 by a (second) UBA-li
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Discussion 161 gradual process requ
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Discussion 163 Although there is no
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Discussion 165 identify mRNAs which
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Human NXFs Discussion 167 While the
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Discussion 169 Therefore, DmNXF1 sh
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References References 171 Aitchison
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References 173 Gadal, O., Strauss,
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References 175 Katahira, J., Strass
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References 177 Murphy, R., Watkins,
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References 179 Strasser, K., Masuda
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Abbreviations Abbreviations 181 ARE
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Curriculum vitae Personal Details N
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Erklärung Hiermit erkläre ich ehr
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